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Abstract:

A biosensor for determining the concentration of an analyte in a
biological sample. The biosensor comprises a support, a reference
electrode or a counter electrode or both disposed on the support, a
working electrode disposed on the support, the working electrode spaced
apart from the other electrode or electrodes on the support, a covering
layer defining a sample chamber over the electrodes, an aperture in the
covering layer for receiving a sample, and at least one layer of mesh in
the sample chamber between the covering layer and the electrodes. The at
least one layer of mesh has coated thereon a silicone surfactant. Certain
silicone surfactants are as effective as fluorinated surfactants with
respect to performance of biosensors. These surfactants, when coated onto
the mesh layer of the biosensor, are effective in facilitating the
transport of aqueous test samples, such as blood, in the sample chamber.

Claims:

1-22. (canceled)

23. A method for making an electrochemical sensor, the method comprising:
applying a working electrode and a counter electrode to the surface of a
substrate; applying a mesh layer on top of the working and counter
electrodes, the mesh layer coated with at least one silicone surfactant,
wherein the at least one silicone surfactant comprises a mixture of
silicone surfactants.

24. The method according to claim 23, wherein the mixture of silicone
surfactants comprises a high molecular weight silicone surfactant and a
low molecular weight silicone surfactant.

25. The method of claim 24, wherein the high molecular weight silicone
surfactant and the low molecular weight silicone surfactant have the
formula: ##STR00006## wherein x ranges from 8 to 9, inclusive, y ranges
from 3 to 4, inclusive, z ranges from 11 to 13, inclusive, and R being
hydrogen for the high molecular weight silicone surfactant; and x is 0, y
is 1, z ranges from 3 to 15, inclusive, and R is hydrogen, methyl, or
acetate for the low molecular weight silicone surfactant.

26. The method of claim 24, wherein the weight fraction of the high
molecular weight silicone surfactant in the mixture ranges from 1% to
99%, inclusive.

27. The method of claim 24, wherein the weight fraction of the low
molecular weight silicone surfactant in the mixture ranges from 1% to 99%
inclusive.

28. The method of claim 23, wherein the method further comprises applying
a working ink on top of the working electrode, the working ink comprising
an analyte responsive enzyme and a mediator.

29. The method of claim 28, wherein the analyte responsive enzyme is
selected from the group consisting of glucose oxidase, glucose
dehydrogenase and 3-hydroxybutyrate dehydrogenase.

30. The method of claim 23, wherein the method comprises applying a
single layer of mesh on top of the working and counter electrodes.

31. The method of claim 23, wherein the method comprises applying two or
more layers of mesh on top of the working and counter electrodes.

32. The method of claim 23, wherein the at least one mesh layer is less
than 150 microns in thickness.

33. The method of claim 23, wherein the method further comprises applying
a cover layer on top of the mesh layer to define an enclosed space over
the electrodes, wherein the mesh layer is positioned in the enclosed
space.

34. A method for making an electrochemical sensor, the method comprising:
applying a working electrode and a counter electrode to the surface of a
substrate; applying a mesh layer on top of the working and counter
electrodes, the mesh layer coated with at least one silicone surfactant,
wherein the at least one silicone surfactant comprises a trisiloxane
surfactant.

35. The method of claim 34, wherein the method further comprises applying
a working ink on top of the working electrode, the working ink comprising
an analyte responsive enzyme and a mediator.

36. The method of claim 35, wherein the analyte responsive enzyme is
selected from the group consisting of glucose oxidase, glucose
dehydrogenase and 3-hydroxybutyrate dehydrogenase.

37. The method of claim 34, wherein the method further comprises applying
a cover layer on top of the mesh layer to define an enclosed space over
the electrodes, wherein the mesh layer is positioned in the enclosed
space.

38. A method for making an electrochemical sensor, the method comprising:
applying a working electrode and a counter electrode to the surface of a
substrate; applying a mesh layer on top of the working and counter
electrodes, the mesh layer coated with at least one silicone surfactant,
wherein the at least one silicone surfactant comprises a cyclosiloxane
surfactant.

39. The method of claim 38, wherein the method further comprises applying
a working ink on top of the working electrode, the working ink comprising
an analyte responsive enzyme and a mediator.

40. The method of claim 39, wherein the analyte responsive enzyme is
selected from the group consisting of glucose oxidase, glucose
dehydrogenase and 3-hydroxybutyrate dehydrogenase.

41. The method of claim 38, wherein the method further comprises applying
a cover layer on top of the mesh layer to define an enclosed space over
the electrodes, wherein the mesh layer is positioned in the enclosed
space.

Description:

REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation application of U.S. patent
application Ser. No. 10/448,643, filed on May 30, 2003, now U.S. Pat. No.
______, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to biosensors. More particularly, this
invention relates to biosensors in which the biological sample is
transported to a sample chamber by means of wicking of fluid.

[0004] 2. Discussion of the Art

[0005] A biosensor is a device for measuring the concentration of an
analyte in a biological sample. A typical biosensor comprises a support,
a reference electrode or a counter electrode or both a reference and a
counter electrode disposed on the support, a working electrode disposed
on the support, the working electrode spaced apart from the other
electrode or electrodes on the support, a covering layer defining an
enclosed space over the electrodes, an aperture in the covering layer for
receiving a sample, and at least one mesh layer in the enclosed space
between the covering layer and the electrodes. The working electrode
includes an enzyme capable of catalyzing a reaction involving a substrate
for the enzyme and a mediator capable of transferring electrons between
the enzyme-catalyzed reaction and the working electrode to create a
current related to the activity of the enzyme and related to the
concentration of the analyte in the sample. Alternatively, instead of an
enzyme, the working electrode can include a substrate capable of
catalyzing a reaction involving an enzyme for the substrate and a
mediator capable of transferring electrons between the
substrate-catalyzed reaction and the working electrode to create a
current related to the activity of the substrate and related to the
concentration of the analyte in the sample. The purpose of the mesh layer
or mesh layers is to define a path for directional flow of the sample
from the aperture through the enclosed space towards the electrodes and
control the height of the enclosed space above the electrodes. The mesh
layers are formed of a woven material and coated with a surfactant. An
example of a biosensor is shown in U.S. Pat. No. 5,628,890, incorporated
herein by reference.

[0006] The test sample is required to be delivered rapidly and uniformly
from a sample application zone, i.e., at the aperture, to a reaction zone
within the enclosed space, which is referred to herein as a sample
chamber. Typically, delivery of the test sample is carried out by wicking
along the mesh layer, which is typically of a hydrophilic character for
biological samples. See U.S. Pat. No. 5,628,890, EP 0170375, U.S. Pat.
No. 5,141,868, and U.S. Pat. No. 6,436,256. Sample chambers of biosensors
are preferably constructed so that they have a small volume for the
purpose of reducing the amount of test sample (generally blood) required
from a patient.

[0007] This approach has advantages in that the use of a mesh layer allows
one dimension of the sample chamber to be tightly controlled while also
reducing the void volume, thereby reducing the volume of the test sample
required. Woven mesh layers are generally fabricated from synthetic
polymeric fibers of known diameter, typically nylon and polyester fibers.
Nylon and polyester fibers are relatively hydrophobic and, consequently,
meshes constructed from the untreated fibers are unsuitable for direct
use for promoting transportation of the test sample in a biosensor.

[0008] U.S. Pat. No. 5,628,890 discloses the use of a surfactant-coated
mesh layer in a biosensor for the purpose of wicking A fluorinated
surfactant, "FLUORAD FC-170C" (3M Company, St. Paul, Minn.) is disclosed
as a preferred surfactant in this system. Manufacture of the fluorinated
surfactant FC-170C was terminated by the 3M Company because of concerns
relating to its effect on the environment. Furthermore, the Environmental
Protection Agency (EPA) has recently imposed restrictions on the
manufacture and use of such surfactants and related substances in the
United States. Similar fluorinated surfactants are still available from
other manufacturers, but there is a legitimate concern that such
materials may be withdrawn from the market in the future.

[0009] Accordingly, the surfactant "FLUORAD FC-170" needs to be replaced
by an equally effective non-fluorinated surfactant, preferably one that
is commercially available. A surfactant must fulfill the following
requirements: long-term stability, ease of applying onto the mesh layer,
in particular, applying by means of an aqueous solution. The Gower
Handbook of Industrial Surfactants lists over 21,000 products.

[0010] Textile spin finishes are non-permanent coatings applied to fibers
and yarns as emulsions in order to improve lubrication and prevent
antistatic build-up during processing (Philip E. Slade, Handbook of Fiber
Finish Technology, Marcel Dekker (1998)). Spreading of the spin finish
emulsion on the surface of the fiber to achieve a uniform coating is
promoted by the addition of surfactants to the formulation. This type of
spreading is somewhat analogous to the situation with respect to
biosensors, where a test sample of high surface tension, i.e., blood, is
applied to a surfactant-coated mesh, where initial wetting occurs
followed by subsequent spreading. However, an important difference is
that the spin finish emulsion contains the surfactant and is applied to
the untreated fiber whereas in the biosensor, the fiber is already coated
with surfactant and a test sample (without surfactant) is applied to the
coated fiber.

[0011] Silicone surfactants are available from a number of manufacturers,
such as, for example, Dow Corning, OSi Specialities, Basildon Chemicals,
Clariant, and Degussa. These surfactants are often used as additives
(minor components) of fiber finishes, which are required during
processing. They are added to finish formulations to promote wetting of
the fiber with the hydrophobic finish and are not used to increase the
hydrophilicity of the finished fiber. The fiber finish is required for
lubrication and anti-static properties during processing. The prior art
offers no specific guidance as to which surfactants will be effective as
spreading agents when applied to mesh in biosensors.

[0012] The mechanics of the spreading/wicking process is complex. The
coating emulsion requires a low surface tension to wet the surface of the
fiber or yarn, but the wicking rate is greater at a high level of surface
tension (Philip E. Slade, Handbook of Fiber Finish Technology, Marcel
Dekker (1998), pg. 45-48). For example, fluorinated surfactants are known
to be among the most effective at lowering surface tension but are
reported to have "a considerable negative effect on wicking" (Wicking of
Spin Finishes and Related Liquids into Continuous Filament Yarns, Y. K.
Kamath, S. B. Hamby, H.-D. Weigmann and M. F. Wilde, Textile Res. J.,
1994, 64, 33-40). This finding is confirmed by spreading studies of
surfactant solutions on Parafilm (K. P. Ananthapadmanabhan, E. D. Goddard
and P. Chandar, Colloids Surf, 1990, 44, 281).

[0013] As stated previously, an important property of a biosensor is its
long-term stability. The biosensor is required to function without any
deterioration in performance for many months after manufacturing.
Satisfactory performance requires the sample chamber to fill rapidly and
uniformly over the shelf life of the product. Given that the coating of
surfactant on the mesh layer is non-permanent and is necessary for
adequate filling of the sample chamber, it follows that the surfactant
itself must be chemically stable, while not undergoing excessive
migration/diffusion from the mesh layer to other surfaces in the
biosensor. Some loss of surfactant from the mesh layer to other
hydrophobic surfaces (such as printed electrode tracks) is considered
beneficial, because these surfaces will become more hydrophilic.
Excessive loss will result in an unacceptable deterioration in wicking
performance, leading ultimately to a catastrophic failure to fill.
Surfactants having high molecular weight, which are either solids or
viscous liquids, are expected to be less mobile and therefore more
capable of providing durable spreading capability. However, such
materials are expected to be less effective as spreading agents than
those surfactants having lower molecular weights.

[0014] It is important to consider the interaction of the surfactant
coated onto the mesh layer with adjacent layers in the biosensor. The
surfactant may inhibit adhesion of other layers to the mesh layer. In
addition, the mesh layer may be adhered to the electrode substrate by a
screen-printed insulating ink, with which the surfactant could interact
adversely. For example, the wet ink printed onto the surfactant-coated
mesh layer may wick along the fibers, resulting in poor print definition.

[0015] It is not a simple case of applying any surfactant (or even
specifically the most effective surfactants such as fluorinated
surfactants) to a mesh layer of a biosensor to achieve rapid and uniform
wicking of the applied test sample. Adequate models of the mechanics and
dynamics of the spreading of surfactant solutions remain to be developed,
largely because the phenomenon is so complex (Silicone Surfactants,
Surfactant Science Series, Vol. 86, ed. Randall M. Hill, Marcel Dekker,
1999, pg. 303-310). Furthermore, there are other critical factors to
consider when selecting a surfactant for specific use in a biosensor;
many of these factors have not been considered previously in the
literature.

[0016] In summary, a number of conflicting factors have to be balanced to
obtain the optimal selection from an enormous range of commercially
available surfactants. These factors include the ability to lower surface
tension, coating stability, coating uniformity, stability of the
surfactant, migration effects, adhesion inhibition effects, wicking
speed, wicking uniformity, toxicity, and printing definition.

SUMMARY OF THE INVENTION

[0017] This invention provides a biosensor for determining the
concentration of an analyte in a biological sample. The biosensor
comprises a support, an arrangement of electrodes disposed on the
support, a covering layer defining an enclosed space over the electrodes,
an aperture in the covering layer for receiving a biological sample, and
at least one mesh layer in the enclosed space between the covering layer
and the electrodes, the at least one mesh layer coated with at least one
silicone surfactant. The arrangement of electrodes preferably comprises a
reference electrode or a counter electrode or both a reference and a
counter electrode disposed on the support, and a working electrode
disposed on the support, the working electrode spaced apart from the
other electrode or electrodes on the support. The working electrode
includes an enzyme capable of catalyzing a reaction involving a substrate
for the enzyme and a mediator capable of transferring electrons between
the enzyme-catalyzed reaction and the working electrode to create a
current related to the activity of the enzyme and related to the
concentration of the analyte in the sample. Alternatively, instead of an
enzyme, the working electrode can include a substrate capable of
catalyzing a reaction involving an enzyme for the substrate and a
mediator capable of transferring electrons between the
substrate-catalyzed reaction and the working electrode to create a
current related to the activity of the substrate and related to the
concentration of the analyte in the sample. The at least one layer of
mesh has coated thereon a silicone surfactant. We have discovered that
certain silicone surfactants are as effective as fluorinated surfactants
with respect to performance of biosensors. These surfactants, when coated
onto the mesh layer of the biosensor, are effective in facilitating the
transport of aqueous test samples, such as blood, from the sample
application zone to the reaction zone in the enclosed space, which is
frequently referred to as a sample chamber. These surfactants are
collectively referred to as silicone surfactants or siloxane surfactants.
These surfactants are preferably non-ionic and may be coated onto a layer
of polymeric mesh, such as, for example, nylon or polyester mesh.

[0018] The silicone surfactants combine a number of properties that are
required for successful use in a biosensor. The overall performance of
the silicone surfactants in the biosensor exceeds that of fluorinated
surfactants, such as "FLUORAD FC-170C." Overall performance is based on
the following parameters: [0019] (a) speed of filling the sample
chamber with the sample; [0020] (b) uniformity of filling the sample
chamber with the sample, i.e., straightness of filling front for the
sample; [0021] (c) shelf life (filling stability), preferably at least 18
months; [0022] (d) minimization of adhesion failure between layers of the
biosensor in contact with the surfactant; [0023] (e) minimization of
seepage of sample between layers of the biosensor; [0024] (f) level of
toxicity, i.e., non-toxicity being preferred; [0025] (g) minimization of
loss in printing definition of the ink layer that holds the mesh layer in
place; [0026] (h) transferability of liquid surfactant by contact to
other surfaces in the biosensor to render them more hydrophilic.

[0027] Silicone surfactants are effective at reducing the surface tension
of aqueous fluids, such as blood. Consequently, hydrophobic meshes coated
with silicone surfactants are capable of being wetted by aqueous fluids,
such as blood.

[0028] Rapid and uniform wicking of blood along the at least one mesh
layer of a biosensor is desired for reproducible results. The time
required to fill a sample chamber of a biosensor containing a mesh layer
coated with a silicone surfactant exceeds that of a sample chamber of a
biosensor containing a mesh layer coated with a fluorinated surfactant,
such as "FLUORAD FC-170C." Wicking uniformity (straightness of moving
liquid front) across mesh coated with silicone surfactants is superior to
that across a mesh coated with a fluorinated surfactant, such as "FLUORAD
FC-170C." Wicking/spreading rates vary according to the structure of the
silicone surfactant. Silicone surfactants having low molecular weight are
very efficient spreading agents, but lack the durability required for a
biosensor. Durability must be balanced with spreading efficiency. For
this reason, mixtures of silicone surfactants having different properties
provide the best overall performance in a biosensor.

[0029] Biosensors having sample chambers containing at least one mesh
layer coated with silicone surfactants have adequate long-term stability.
The sample chambers continue to fill rapidly and uniformly for at least
18 months when stored at 30° C. A shelf life of longer than 18
months is not required for a biosensor. Long-term stability is desired so
that a catastrophic failure is not observed near the end of shelf life.

[0030] Silicone surfactants are non-toxic and do not irritate the skin. In
contrast, fluorinated surfactants are toxic. Silicone surfactants are
freely available from a number of suppliers.

[0031] In some biosensor systems the surfactant-coated mesh layer is
covered with a polymeric film to form the sample chamber. Good adhesion
between the mesh layer/insulating layer and the polymeric film is
important to ensure that the sample chamber remains intact and to specify
the volume of the test sample required to fill the sample chamber. If
adhesion were poor, the polymeric film could peel away or seepage of the
test sample may occur between the polymeric film and the mesh
layer/insulating layer at the edge of the sample chamber. Such seepage
will increase the volume of sample required to fill the sample chamber.
Silicone surfactants have no adverse effect on the adhesion between the
layers forming the sample chamber.

[0032] In some biosensors, the surfactant-coated mesh layer is held in
place by overprinting with a layer of insulating ink. The surfactant
coating may promote wicking of the wet insulating ink along the fibers of
the mesh layer, leading to a poor print definition. In extreme cases, the
insulating ink could cover areas that are required to be exposed.
Silicone surfactants are comparable to fluorinated surfactants such as
"FLUORAD FC-170C" in providing satisfactory definition of the insulating
ink layer in the biosensor, when applied to the mesh layer at an
equivalent level.

[0033] The biosensors of this invention employ non-toxic and
environmentally friendly silicone surfactants in place of fluorinated
surfactants (e.g., "FLUORAD FC-170." The silicone surfactants act as
wetting agents when applied to polymeric meshes, such as polyamide (e.g.,
nylon) and polyester (e.g., PET). The hydrophobic polyester and polyamide
meshes, when coated with silicone surfactants, become hydrophilic, and
hence promote the lateral transport/flow/wicking of an aqueous sample,
such as blood, from a sample application zone to a reaction zone in a
diagnostic assay device, such as a biosensor.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1 is an exploded perspective view of a biosensor suitable for
use in this invention.

[0035]FIG. 2 is a series of video images (individual frames) showing
water wicking into sample chambers of a model biosensor.

[0036] FIG. 3 is a series of video images (individual frames) showing
blood wicking into sample chambers of a model biosensor.

[0037] FIG. 4 is a series of video images (individual frames) showing
blood wicking into sample chambers of a manufactured biosensor.

[0038] FIG. 5 is a graph showing coated weight of surfactant as a function
of concentration of surfactant in coating solution.

[0039]FIG. 6 is a graph showing coated weight of surfactant as a function
of concentration of surfactant in coating solution.

[0040]FIG. 7 is a graph showing coated weight of surfactant as a function
of concentration of surfactant in coating solution.

[0041] FIG. 8A is a graph showing percentage of biosensors that would fill
as a function of storage time and storage temperature.

[0042] FIG. 8B is a graph showing percentage of biosensors that would fill
as a function of storage time and storage temperature.

[0043] FIG. 9 is a graph showing shelf life of biosensors at a given
storage temperature as a function of concentration of surfactant in
coating bath.

[0044] FIG. 10A is a graph showing percentage of sample chambers of
biosensors that would fill as a function of storage time and storage
temperature.

[0045] FIG. 10B is a graph showing the time to fill the sample chambers of
biosensors as a function of storage time and storage temperature.

[0046]FIG. 11A is a graph showing percentage of sample chambers of
biosensors that would fill as a function of storage time and storage
temperature.

[0047] FIG. 11B is a graph showing time to fill the sample chambers of
biosensors as a function of storage time and storage temperature.

[0048] FIG. 12 is a contour plot showing variation in time of filling of a
biosensor having a single mesh layer as a function of both the type of
surfactant used and the storage time, where storage temperature was
50° C.

[0049] FIG. 13A are video images showing sample chambers of biosensors
having two mesh layers filled with blood. The biosensors were stored at
ambient temperature and the surfactants were DC 193 and FSN-100.

[0050]FIG. 13B are video images showing sample chambers of biosensors
having two mesh layers filled with blood. The biosensors were stored at
40° C. for four weeks and the surfactants were DC 193 and FSN-100.

DETAILED DESCRIPTION OF THE INVENTION

[0051] As used herein, the terms "silicone" and "siloxane" are synonymous.
The term "silicone" denotes a synthetic polymer
(RnSiO.sub.(4-n)/2)m, where n=1 to 3, inclusive and m is equal
to or greater than 2. A silicone contains a repeating silicon-oxygen
backbone and has organic groups R attached to a significant proportion of
the silicon atoms by silicon-carbon bonds. In commercial silicones most R
groups are methyl; longer alkyl, fluoroalkyl, phenyl, vinyl, and a few
other groups are substituted for specific purposes. Some of the R groups
can also be hydrogen, chlorine, alkoxy, acyloxy, or alkylamino, etc.
These polymers can be combined with fillers, additives, and solvents to
result in products classed as silicones. See Kirk-Othmer Encyclopedia of
Polymer Science and Technology, Volume 15, John Wiley & Sons, Inc. (New
York: 1989), pages 204-209, 234-265, incorporated herein by reference.

[0052] This invention provides a biosensor for determining the
concentration of an analyte in a biological sample. The biosensor
comprises a support, a reference electrode or a counter electrode or both
a reference and a counter electrode disposed on the support, a working
electrode disposed on the support, the working electrode spaced apart
from the other electrode or electrodes on the support, a covering layer
defining a sample chamber over the electrodes, an aperture in the
covering layer for receiving a sample, and at least one layer of mesh in
the sample chamber between the covering layer and the electrodes on the
support. The working electrode includes an enzyme capable of catalyzing a
reaction involving a substrate for the enzyme and a mediator capable of
transferring electrons between the enzyme-catalyzed reaction and the
working electrode to create a current related to the activity of the
enzyme and related to the concentration of the analyte in the sample.
Alternatively, instead of an enzyme, the working electrode can include a
substrate capable of catalyzing a reaction involving an enzyme for the
substrate and a mediator capable of transferring electrons between the
substrate-catalyzed reaction and the working electrode to create a
current related to the activity of the substrate and related to the
concentration of the analyte in the sample. The at least one layer of
mesh has coated thereon at is least one silicone surfactant.

[0053] By applying certain silicone surfactants to the at least one layer
of mesh, which is typically constructed of a hydrophobic polymeric
material, the at least one layer of mesh is rendered hydrophilic and can
be wetted by water and water-borne solutions such as blood. This treated
mesh facilitates the transport/flow/wicking of aqueous reagents, such as
blood, to a reaction zone covered by the at least one layer of mesh,
where the active ingredients of the biosensor are located for a
quantitative assay. Without aid of the at least one surfactant, it would
be impossible to wet the reaction zone by the reagents alone.

[0054] Representative examples of classes of silicone surfactants suitable
for use in this invention are illustrated below:

##STR00001##

##STR00002##

##STR00003##

##STR00004##

[0055] The silicone surfactants that are preferred for use in this
invention have the general formula:

[0057] R represents hydrogen, an alkyl group, preferably having 1 to 4
carbon atoms, or an alkyl ester group, preferably having 1 to 4 carbons
atoms in the alkyl portion, where, if y>1, each R can be the same or
different on any given molecule, with R preferably being hydrogen or
methyl;

[0058] x represents 0 up to a value in combination with y and z where the
surfactant is not a liquid, x preferably being zero for the trisiloxane
surfactants useful in surfactant mixtures;

[0059] y represents 1 up to a value in combination with x and z where the
surfactant is not a liquid.

[0060] In general, non-ionic silicone surfactants comprise two structural
units, a silicone group and a polyalkylene oxide chain; non-ionic
silicones are preferred over cationic, anionic, or amphoteric silicones,
because non-ionic surfactants allow the covering tape to readily adhere
to the layer of coated mesh. The polyalkylene oxide chain preferably
comprises ethylene oxide (EO) units or propylene oxide (PO) units or a
mixture of the two. Varying the polyalkylene chain length and EO/PO ratio
varies the properties of the surfactants. For example, increased water
solubility is seen for those surfactants having a high ratio of EO to PO.
In addition, surfactants having a short polyalkylene chain are known to
be "superwetting agents." Furthermore, surfactants having high molecular
weight are viscous liquids, which have good stability when coated onto
polymeric fibers, due to a low mobility.

[0061] Surfactants that are particularly suitable for this invention have
the following characteristics: [0062] (a) Surface tension range: 20 to
35 mN/m [0063] (b) Estimated viscosity range: 1 to 5000 cSt [0064] (c)
Estimated specific gravity range: 0.98 to 1.20 [0065] (d) Solubility or
dispersibilty in water and alcohols: up to 10%. [0066] (e) Cloud point:
preferably >25° C. at 10% concentration in coating solution of
choice (water/alcohol mixture) [0067] (f) Molecular weight: number
average molecular weight based on number of surfactants ranges from about
500 to about 30,000 [0068] (g) Additives: no additives are deliberately
added beyond the manufacturer's formulation, which may contain additives
or stabilizers or both [0069] (h) Surfactant should be a liquid coating
that can transfer to other surfaces by contact

[0070] The silicone surfactant preferred for this invention has the trade
name Dow Corning 193 Fluid, because it has a good balance of aqueous
solubility, wetting ability, and stability. This silicone surfactant has
a preferred rake-type structure (see the structures shown in Formulas I
and V) and contains only EO with no PO. Dow Corning 193 Fluid has a
molecular weight of approximately 2500.

[0071] The choice of a single surfactant for coating fibers of a mesh
layer requires a compromise between various conflicting factors. However,
a mixture of surfactants applied to the fibers of a mesh layer may
achieve an enhanced effect, because a combination of "superwetting" agent
and high molecular weight products can be used. A "superwetting" agent
alone will have poor stability, and a surfactant having very high
molecular weight will have poor wetting properties and aqueous
solubility, but good stability.

[0072] The surfactant or mixture of surfactants is first dissolved in an
organic is solvent (e.g., acetone), water, or a mixture of water and
organic solvent (e.g., water/isopropanol) to yield a solution having a
concentration in the range 0.01 to 10%, based on weight. The polymeric
meshes are typically in a roll format and have various dimensions.
Coating of the surfactant is achieved by continuously transporting the
polymeric mesh from one end to the other through a bath of the solution
of surfactant at a constant speed. A drying process is used to remove the
solvent from the coating composition. In the drying process, temperatures
of up to 130° C. can be used.

[0073] Silicone surfactants are environmentally friendly and non-toxic, as
compared with fluorinated surfactants, such as "FLUORAD FC-170C", which
are toxic and persistent in the environment. Silicone surfactants are at
least equivalent to the fluorinated surfactants currently available, with
respect to performance of current biosensor strips. The sample chambers
fill rapidly and uniformly with blood over the shelf life of the strips.
Silicone surfactants are manufactured by many companies; hence, there is
no problem of shortage of suppliers, in contrast to fluorinated
surfactants.

[0074] A stable, rapidly-fillable sample chamber containing a
surfactant-coated mesh system for a biosensor can be obtained where
through the use of a single liquid silicone surfactant of Formula V,
where x ranges from 7 to 12, inclusive, y ranges from 3 to 5, inclusive,
z ranges from 3 to 15, inclusive, R being hydrogen or methyl. Preferably,
x ranges from 8 to 9, inclusive, y ranges from 3 to 4, inclusive, z
ranges from 11 to 13, inclusive, R being hydrogen. A silicone surfactant
having these values of x, y, and z is similar to Dow Corning 193 Fluid.
Commercial surfactants typically have a range of x, y, and z values,
whereby a distribution of molecular weights is obtained.

[0075] A stable, rapidly fillable sample chamber containing a
surfactant-coated mesh system for a biosensor can be obtained through the
use of a mixture of two silicone surfactants. Preferably, the mixture
comprises a surfactant having high molecular weight having satisfactory
properties with respect to stability of the biosensor and a surfactant
having low molecular weight having satisfactory properties for rapid
filling of the sample chamber. Most preferably, in the surfactant of high
molecular weight (Formula V), x ranges from 8 to 9, inclusive, y ranges
from 3 to 4, inclusive, z ranges from 11 to 13, inclusive, R being H,
i.e., similar to Dow Corning 193 Fluid. Most preferably, in the
surfactant having low molecular weight (Formula V), preferably a
trisiloxane surfactant, x is 0, y is 1, z ranges from 3 to 15, inclusive,
R is hydrogen, methyl, or acetate.

[0076] The weight fraction of the silicone surfactant having high
molecular weight in the mixture preferably ranges from 1% to 99%,
inclusive. The weight fraction of the silicone surfactant having low
molecular weight in the mixture preferably ranges from 1% to 99%,
inclusive. The most preferred weight fraction for the silicone surfactant
having high molecular weight ranges from 50% to 99%, inclusive, and the
most preferred weight fraction for the silicone surfactant having low
molecular weight ranges from 1% to 50%, inclusive.

[0077] The coating weight of the surfactant on the layer of mesh
preferably ranges from about 0.01% to about 8%, based on the weight of
the mesh.

[0078] Preferably, a mixture of surfactants suitable for the present
invention will have two components as described previously. However,
mixtures of surfactants containing three or more silicone surfactants can
be used to achieve optimal effects.

[0079] A biosensor strip 10 suitable for this invention is illustrated in
FIG. 1. Referring to FIG. 1, an electrode support 11, preferably an
elongated strip of polymeric material (e.g., polyvinyl chloride,
polycarbonate, polyester, or the like) supports three tracks 12a, 12b,
and 12c of electrically conductive ink, preferably comprising carbon.
These tracks 12a, 12b, and 12c determine the positions of electrical
contacts 14a, 14b, and 14c, a reference electrode 16, a working electrode
18, and a counter electrode 20. The electrical contacts 14a, 14b, and 14c
are insertable into an appropriate measurement device (not shown). This
type of biosensor is shown in U.S. Ser. No. 10/062,313, filed Feb. 1,
2002, now U.S. Pat. No. 6,863,800, incorporated herein by reference.
While this illustration involves a biosensor having a working electrode,
a reference electrode, and a counter electrode, it is not critical for a
biosensor to have three electrodes. One electrode can be used to perform
the functions of the reference electrode and the counter electrode.
Auxiliary electrodes can be added for other purposes. What is required is
an electrode arrangement comprising at least a working electrode and a
reference electrode. A type of biosensor having a working electrode and a
single electrode to perform the functions of the reference electrode and
the counter electrode is shown in WO 99/19507, published 22 Apr. 1999,
incorporated herein by reference.

[0080] Referring again to FIG. 1, each of the elongated portions of the
conductive tracks 12a, 12b, and 12c can optionally be overlaid with a
track 22a, 22b, and 22c of conductive material, preferably made of a
mixture comprising silver particles and silver chloride particles. The
enlarged exposed area of track 22b overlies the reference electrode 16. A
layer of a hydrophobic electrically insulating material 24 further
overlies the tracks 22a, 22b, and 22c. The positions of the reference
electrode 16, the working electrode 18, the counter electrode 20, and the
electrical contacts 14a, 14b, and 14c are not covered by the layer of
hydrophobic electrically insulating material 24. This hydrophobic
electrically insulating material 24 serves to prevent short circuits. The
layer of hydrophobic electrically insulating material 24 has an opening
26 formed therein. This opening 26 provides the boundary for the reaction
zone of the biosensor strip 10. Because this insulating material is
hydrophobic, it can cause the sample to be restricted to the portions of
the electrodes in the reaction zone. The working electrode 18 comprises a
layer of a non-reactive electrically conductive material on which is
deposited a layer 28 containing a working ink for carrying out an
oxidation-reduction reaction. At least one layer of mesh 30 overlies the
electrodes. This mesh layer 30 protects the printed components from
physical damage. The mesh layer 30 also helps the sample to wet the
electrodes by reducing the surface tension of the sample, thereby
allowing it to spread evenly over the electrodes. A cover 32 encloses the
surfaces of the electrodes that are not in contact with the electrode
support 11. This cover 32 is a liquid impermeable membrane. The cover 32
includes a small aperture 34 to allow access of the applied sample to the
underlying mesh layer 30.

[0081] The layer of working ink 28 is deposited on that portion of the
electrically conductive material of the working electrode 18 where the
oxidation-reduction reaction is to take place when a sample is introduced
to the biosensor strip 10. The layer of the working ink 28 can be applied
to the working electrode 18 as a discrete area having a fixed length. The
working ink comprises reagent(s) that are responsive to the analyte of
interest deposited on the non-reactive electrically conductive material.
As used herein, the term "reagent(s)" means at least one reagent. Typical
analytes of interest include, for example, glucose and ketone bodies.
Typical non-reactive electrically conductive materials include, for
example, carbon, platinum, palladium, and gold. A semiconducting material
such as indium doped tin oxide can be used as the non-reactive
electrically conductive material. In preferred embodiments, the working
ink comprises a mixture of an oxidation-reduction (redox) mediator and an
enzyme. Alternatively, instead of an enzyme, the working ink can contain
a substrate that is catalytically reactive with an enzyme to be assayed.
For example, when the analyte to be measured is glucose in blood, the
enzyme is preferably glucose oxidase, and the redox mediator is
preferably ferrocene or a derivative thereof. Other mediators that are
suitable for use in this invention include a ferricyanide salt and a
phenanthroline quinone or a derivative thereof. In the biosensor strips
of this invention, the reagent(s) are preferably applied in the form of
ink containing particulate material and having binder(s), and,
accordingly, does not dissolve rapidly when subjected to the sample. In
view of this feature, the oxidation-reduction reaction will occur at the
interface of working electrode 18 and the sample. The glucose molecules
diffuse to the surface of the working electrode 18 and react with the
enzyme/mediator mixture.

[0082] In addition to being applied to the working electrode 18, a layer
of the working ink can be applied to any of the other electrodes, when
desired, as a discrete area having a fixed length.

[0083] The thickness of the layer of non-reactive electrically conductive
material is determined by the method of applying the layer. In the case
of a layer deposited by printing, e.g., screen-printing, the thickness of
the layer typically ranges from about 10 micrometers to about 25
micrometers. In the case of a layer deposited by vapor deposition, the
thickness of the layer typically ranges from about less than 1 micrometer
to about 2 micrometers. The layer of the working ink 28 that has been
deposited on the working electrode 18 typically has a dry thickness of
from about 2 to about 50 micrometers, preferably from about 10 to about
25 micrometers. The actual dry thickness of the deposited layer of the
working ink 28 will depend to some extent upon the technique used to
apply the working ink. For example, a thickness of from about 10 to about
25 micrometers is typical for a layer of working ink applied by means of
screen-printing.

[0084] The reference electrode 16 is typically formed by screen-printing a
mixture comprising a mixture of silver and silver chloride on the
electrode substrate 11. For materials to which such a mixture does not
readily adhere, it is preferred to deposit a layer of carbon on the
electrode support to act as a primer layer for the mixture. This mixture
is often referred to as ink. The mixture typically has a carrier
comprising an organic solvent. Alternatives to the mixture of silver and
silver chloride include mixtures of Ag and AgBr, mixtures of Ag and AgI,
and mixtures of Ag and Ag2O. The printed layer associated with the
reference electrode 16 extends to partially cover the track of the carbon
layer associated with the reference electrode 16, where the printed layer
extends into the reaction zone. It is preferred to cover parts of the
tracks 12a, 12b, and 12c outside the reaction zone with the mixture of
silver and the silver compound associated therewith, so that the total
electrical resistance of each track is reduced. Because no current flows
through the reference electrode 16, non-classical reference electrodes
can be used as the reference electrode. These non-classical electrodes
can be formed either by simply employing a conductive material, such as,
for example, carbon, platinum, or palladium, as the reference electrode
or by having the working ink deposited on the conductive material that
forms the reference electrode. The reference electrode 16 preferably has
equal or smaller dimensions compared to those of the working electrode
18.

[0085] If the working ink is deposited on a conductive material to form
the reference electrode 16, the reagent(s) are deposited only on the
portion of the electrode that is in the reaction zone to minimize the
electrical resistance of the track 12c.

[0086] In the case of carbon being deposited to form the reference
electrode 16 (i.e., an electrically conductive electrode without
oxidation-reduction reagents), no additional material is required to be
deposited on the surface of the reference electrode. The carbon can be
doped with metal particles to increase the conductivity of the carbon.

[0087] Any electrically conductive material can be used to form the
counter electrode 20. Preferred materials for forming the counter
electrode 20 include, but are not limited to, platinum, palladium,
carbon, gold, silver, and mixtures of silver and silver chloride (as in
the reference electrode 16). In another embodiment, reagent(s) that form
the working ink can be deposited on the conductive material of the
counter electrode 20. If the working ink is deposited on a conductive
material to form the counter electrode 20, the reagent(s) are deposited
only on the portion of the electrode that is in the reaction zone to
minimize the electrical resistance of the track 12b.

[0088] The dimensions of the counter electrode 20 are preferably equal to
or greater than those of the reference electrode 16. It is preferred that
the counter electrode 20 be of a size equal to or greater than the
working electrode 18, though this preference is not required at low
levels of current. In functional terms, the size of the reference
electrode is not critical; the size of the working electrode is selected
on the basis of signal to noise ratio desired; the size of the counter
electrode is selected to minimize resistance to current flow.

[0089] The counter electrode 20 must be in electrical contact with the
working electrode 18 during the measurement. When current flows through
the counter electrode 20, the flow of electrons produces an
electrochemical reaction (a reduction reaction) sufficient to allow the
electrons to flow. The counter electrode 20 must be positioned at a
sufficient distance from the working electrode 18 so that the reactive
species generated at the counter electrode 20 do not diffuse to the
working electrode 18.

[0090] The reaction zone can have total area ranging from about 1 mm2
to about 20 mm2, preferably about 5 mm2. The area of the
working electrode typically ranges from about 0.5 to about 5 mm2,
preferably about 1.0 mm2. The reference electrode and the counter
electrode typically have areas ranging from about 0.2 to about 4.0
mm2, preferably about 0.5 mm2.

[0091] The biosensor strip 10 typically has a width of from about 4.5 to
about 6.5 mm. The electrode support 11 can be made from any material that
has an electrically insulating surface, such as, for example, polyvinyl
chloride, polycarbonate, polyester, paper, cardboard, ceramic,
ceramic-coated metal, and blends of these materials (e.g., a blend of
polycarbonate and polyester).

[0092] Electrically conductive material can be applied to the electrode
support 11 by a deposition method such as screen-printing. This deposit
of electrically conductive material forms the contact areas 14a, 14b, and
14c, which areas allow the analyte monitor to interface with the
biosensor strip 10. The conductive material further provides electrical
connections between the contact areas and the active reagent(s) deposited
on the electrode(s) of the biosensor strip 10. The formulation for the
electrically conductive material can be an air-dryable composition
comprising carbon dispersed in an organic solvent. Alternative
formulations include carbon dispersed in an aqueous solvent. Alternative
electrically conductive materials that can be used in place of carbon
include, but are not limited to, such materials as silver, gold,
platinum, and palladium. Other methods of drying or curing the
formulations containing the electrically conductive material include the
use of infrared radiation, ultraviolet radiation, and radio frequency
radiation. In an alternative method of application, the electrically
conductive material can be deposited by means of a vapor deposition
technique.

[0093] As stated previously, inks suitable for use in this invention can
be screen-printed. Other ways of depositing the inks include drop
coating, inkjet printing, volumetric dosing, gravure printing,
flexographic printing, and letterpress printing. The electrically
conductive portions of the electrodes are preferably screen-printed or
deposited by means of sputtering or vapor deposition techniques. The
reagents are preferably deposited by screen-printing or drop coating the
formulations on the surface of the electrically conductive portion of the
electrode. In the case of screen-printing, the reagents can be converted
into particulate form wherein the particles contain carbon or silica,
with carbon being preferred. In the drop coating formulation, the
reagents can be mixed with a polymer (such as, for example, carboxy
methyl cellulose, hydroxy ethyl cellulose, polyvinyl alcohol, etc.)
solution to obtain a viscous solution, which is then dispensed on the
area of interest. The inks can further include a polysaccharide (e.g., a
guar gum, an alginate, locust bean gum, carrageenan, or xanthan), a
hydrolyzed gelatin, an enzyme stabilizer (e.g., glutamate or trehalose),
a film-forming polymer (e.g., a polyvinyl alcohol, hydroxyethyl
cellulose, polyvinyl pyrrole, cellulose acetate, carboxymethyl cellulose,
and poly(vinyl oxazolidinone), a conductive filler (e.g., carbon), a
defoaming agent, a buffer, or combinations of the foregoing. Other
fillers for the inks include, but are not limited to, titanium dioxide,
silica, and alumina.

[0094] It is preferred that the length of the path to be traversed by the
sample (i.e., the reaction zone) be kept as short as possible in order to
minimize the volume of sample required. With respect to the biosensor
strip described herein, the volume of sample required is preferably no
greater than 5 microliters, and more preferably ranges from about 0.5
microliters to about 2.5 microliters. The maximum length of the reaction
zone can be as great as the length of the biosensor strip. However, the
corresponding increase in resistance of the sample limits the length of
the reaction zone to a distance that allows the necessary response
current to be generated. Positioning the electrodes in the manner
described herein has the further advantage of preventing completion of
a-circuit (and thus preventing detection of a response current) before
the working electrode 18 has been completely covered by the sample.

[0095] As shown in FIG. 1, a mesh layer 30 overlies the electrodes. As
stated previously, this mesh layer 30 protects the printed components
from physical damage, and the mesh layer 30 also helps the sample to wet
the electrodes by reducing the surface tension of the sample, thereby
allowing it to spread evenly over the electrodes. Preferably, this mesh
layer 30 extends over the entire length of the reaction zone, between and
including the position at which the sample is introduced and the region
where the electrodes are disposed. Preferably, this mesh layer 30 is
constructed of woven strands of polyester. Alternatively, any woven or
non-woven material can be used, provided that it does not occlude the
surface of the electrode such that normal diffusion of the sample is
obstructed. The thickness of the mesh is selected so that the depth of
the sample is sufficiently low that a high sample resistance is produced.
Preferably, the mesh layer 30 is not more than 150 μm in thickness.
Preferably, the mesh layer 30 has a percent open area of about 35% to
about 45%, a fiber count of about 40 per cm to about 60 per cm, a fiber
diameter of about 70 μm to about 100 μm, an average mesh opening of
about 67 to about 180 μm, and a thickness of from about 100 μm to
about 160 μm. The diameter of the fiber can be outside the preferred
range, e.g., about 10 μm to about 1000 μm. A particularly preferred
mesh is PE130 HD mesh, available from Sefar (formerly ZBF), CH-8803,
Ruschlikon, Switzerland.

[0096] The mesh layer 30 is coated with a surfactant. A surfactant coating
is necessary only if the material of the mesh layer 30, itself, is
hydrophobic (for example, nylon or polyester). If the material of the
mesh layer 30 is hydrophilic, the surfactant coating can be used or can
be omitted. A surfactant loading of from about 15 to about 20 μg/mg of
mesh is preferred for most applications. The preferred surfactant loading
will vary depending on the type of mesh layer and surfactant used and the
sample to be analyzed. The preferred surfactant loading can be determined
empirically by observing flow of the sample through the mesh layer 30
with different levels of surfactant.

[0097] The mesh layer 30 can be held in place by the layer of hydrophobic
electrically insulating material 24. This layer of electrically
insulating material 24 is preferably applied by screen-printing the ink
over a portion of the periphery of the mesh layer 30. Together, the mesh
layer 30 and the layers of hydrophobic electrically insulating material
24 surround and define a reaction zone 30 suitable for the sample to
travel from the position at which the sample is introduced at one end of
the strip towards the reference electrode 16, then toward the working
electrode 18, and then toward the counter electrode 20. The hydrophobic
electrically insulating material 24 impregnates the mesh layer 30 outside
of the reaction zone 30. The hydrophobic electrically insulating material
24 thus defines the reaction zone 30 by preventing the sample from
infiltrating the portions of the mesh layer 30 covered by the layers of
hydrophobic electrically insulating material 24. A hydrophobic
electrically insulating material 24 preferred for impregnating the mesh
layers is "SERICARD" (Sericol, Ltd., Broadstairs, Kent, UK). Another
preferred hydrophobic electrically insulating material is commercially
available as "POLYPLAST" (Sericol Ltd., Broadstairs, Kent, UK).

[0098] A layer of dielectric ink can optionally be applied to cover the
majority of the printed carbon and silver/silver chloride tracks. In this
case, two areas are left uncovered, namely the electrical contact areas
and the sensing areas in the reaction zone. This layer of dielectric ink
serves to define the area constituting the reaction zone, and to protect
exposed tracks from short circuit.

[0099] As shown in FIG. 1, a cover 32 encloses the surfaces of the
electrodes that are not in contact with the electrode support 11. The
cover 32 is a liquid impermeable membrane. This cover 32 can be a
flexible tape made of polyester or similar material. The cover 32
includes a small aperture 34 to allow access of the applied sample to the
underlying mesh layer 30. This cover 32 encloses the exposed surfaces of
the working electrode 18, the reference electrode 16, and the counter
electrode 20. Thus, the cover 32 maintains the available sample space
over the electrodes at a fixed depth, which is equivalent to the
thickness of the mesh layer 30. The positioning of this cover 32 ensures
that the resistance of the sample is maintained at a high level.

[0100] The aperture 34 is positioned to overlie an end of the mesh area
upstream of the reference electrode 16, such that the exposed mesh area
beneath the aperture 34 can be used as a point of access or application
for a liquid sample, whereby the sample contacts the reference electrode
16 before the sample contacts the working electrode 18 and the counter
electrode 20. Of course, the aperture 34 must overlie an end of the mesh
area that is not covered by the hydrophobic electrically insulating ink
30. The size of this aperture 34 is not critical, but it should be
sufficiently large to allow sufficient volume of sample to pass through
to the mesh layer 30. The aperture 34 should not be so large as to allow
any portion of the liquid sample to contact any of the electrodes before
contacting the mesh layer 30. The aperture 34 can be formed in the liquid
impermeable cover 32 by any suitable method (e.g., die punching).

[0101] The liquid impermeable cover membrane 32 can be affixed to the
biosensor strip by means of a suitable method of adhesion. Preferably,
affixing is achieved by coating the underside of the flexible tape with a
layer of hot melt glue, and then heat welding the tape to the surface of
the layer of hydrophobic electrically insulating ink 24. The layer of hot
melt glue typically has a coating weight of from about 10 to about 50
g/m2, preferably from about 20 to about 30 g/m2. Pressure
sensitive adhesives or other equivalent methods of adhesion may also be
used. Care should be taken when the tape is applied, because the heat and
pressure applied to the tape layer can melt the "SERICARD" ink and can
cause it to smear onto adjoining areas. Care should also be taken so that
the tape does not cover the electrodes, the reaction zone, or the area
where the sample is applied.

[0102] The upper surface of the liquid impermeable cover 32 can also be
provided with a layer of silicone or other hydrophobic material. This
additional layer serves to drive the applied sample onto the portion of
exposed mesh layer 30 at the sample application point, thereby rendering
the application of small volumes of sample much simpler.

[0103] In use, a biosensor strip 10 of this invention is connected, via
electrode contacts 14a, 14b, and 14c, to a measuring device (not shown).
A liquid sample is applied through aperture 34, and the sample moves
along the reaction zone. The progress of the sample is sufficiently
impeded by the mesh layer 30, thereby allowing the sample to form a
uniform flow front. Air is displaced through the upper portion of the
mesh layer 30 to and through the aperture 34. The sample first completely
covers the working electrode 18 and the reference electrode 16, and only
then approaches and covers and the counter electrode 20, thereby
completing the circuit and causing a response to be detected by the
measuring device.

[0104] Measuring devices that are suitable for use in this invention
include any commercially available analyte monitor that can accommodate a
biosensor strip having a working electrode, a reference electrode, and a
counter electrode. Such analyte monitors can be used to monitor analytes,
such as, for example, glucose and ketone bodies. In general, such a
monitor must have a power source in electrical connection with the
working electrode, the reference electrode, and the counter electrode.
The monitor must be capable of supplying an electrical potential
difference between the working electrode and the reference electrode of a
magnitude sufficient to cause the electrochemical oxidation of the
reduced mediator. The monitor must be capable of supplying an electrical
potential difference between the reference electrode and the counter
electrode of a magnitude sufficient to facilitate the flow of electrons
from the working electrode to the counter electrode. In addition, the
monitor must be capable of measuring the current produced by the
oxidation of the reduced mediator at the working electrode.

[0105] In this invention, the liquid sample is preferably a sample of
whole blood. Alternatively, the liquid sample can be whole blood that has
been filtered or treated to remove red blood cells or other hemocytes.
Other biological samples, such as, for example, plasma, serum, urine,
saliva, interstitial fluid, can be used.

[0106] The following non-limiting examples further illustrate this
invention.

[0108] The purpose of this example was to qualitatively assess the
spreading ability of water onto a nylon mesh coated with a surfactant.

[0109] Various types of surfactants (TABLE 2) were coated onto strips of
nylon mesh (15 mm wide, NY151, Sefar, Switzerland). Coating was performed
by immersing the mesh in an aqueous acetone solution containing the given
surfactant (0.1% w/w). The immersed mesh was then slowly withdrawn from
the solution and dried in an oven at a temperature of 50° C. for
two days. Two nylon mesh control samples, one uncoated and one coated
with the surfactant FC-170C, were employed.

[0110] Short lengths of mesh coated with the various surfactants were
sandwiched between a polyester sheet and a transparent polyester film by
means of a tape having both major surfaces thereof coated with an
adhesive (double-sided adhesive tape). This arrangement provided a model
sample chamber with a height of approximately 180 to 190 μm, as
measured by a micrometer.

[0111] Colored water (10 μl) was applied by automatic pipette (Gilson)
to the edge of the model sample chamber. The progress of the water as it
was drawn into the sample chamber was recorded by a high-speed video
camera at a speed of 16 frames per second. Examples of captured video
images are shown in FIG. 2. The small number in the upper left corner of
each frame represents the number of the frame from the introduction of
the sample. The surfactant used was G2109. No spreading of water in the
model sample chamber was seen for the uncoated control and the mesh
coated with surfactants S-20, PP822, and FSO-100. These three surfactants
were not included in further examples. In contrast, water did wick into
model sample chambers containing mesh coated with surfactants FC-170C
(control), G2109, PP3, DC 193, DC 190, and FSN-100.

[0112] The purpose of this example was to qualitatively assess the
spreading ability of blood onto a nylon mesh coated with a surfactant.

[0113] Model sample chambers were constructed using nylon mesh from
Example 1 that had been coated with the surfactants that were successful
in spreading water (TABLE 3). Again, two nylon mesh control samples, one
uncoated and one coated with the surfactant FC-170C, were employed.

[0114] Short lengths of mesh coated with the various surfactants were
sandwiched between a polyvinyl chloride (PVC) sheet overprinted with a
silver/silver chloride ink and a transparent polyester film by means of
double-sided adhesive tape. This arrangement provided a model sample
chamber having a height of approximately 180 μm, as measured by a
micrometer. The use of a hydrophobic silver/silver chloride layer on the
surface of one side of the model sample chamber was intended to reproduce
more closely the environment within a typical commercially available
sample chamber.

[0115] Freshly drawn venous blood (10 μl) was applied by automatic
pipette (Gilson) to the edge of the model sample chamber. The progress of
the blood as it was drawn into the sample chamber was recorded by a
high-speed video camera at a speed of 16 frames per second. Examples of
captured video images are shown in FIG. 3. The small number in the upper
left corner of each frame represents the number of the frame from the
introduction of the sample. The surfactant used was G2109. No spreading
of blood in the model sample chamber was seen for the uncoated control.
Blood did wick into model sample chambers containing mesh coated with
surfactants FC-170C (control), G2109, PP3, DC 193, DC 190, and FSN-100.
However, the wicking speed for blood was observed to be slower than that
recorded for water in Example 1. This result is expected because of the
higher viscosity of blood relative to that of water.

[0116] The purpose of this example was to qualitatively assess the
spreading ability of blood onto a nylon mesh coated with surfactants,
where the nylon mesh is incorporated into a biosensor having two layers
of mesh.

[0117] Biosensors substantially similar to those described in U.S. Pat.
No. 5,628,890, incorporated herein by reference, were constructed. The
biosensors contained a working electrode, an electrode that performs as a
reference electrode and a counter electrode, and a trigger electrode. The
sample chamber of the biosensor contained two layers of nylon mesh, both
of which were coated with surfactant. The layers of nylon mesh were
designated NY64 and NY151, both supplied by Sefar (Switzerland). A
dip-coating technique was used to coat separate rolls of each type of
mesh with the various surfactants being evaluated. TABLE 4 shows the
identity and amount of surfactant coated on each layer of mesh.

[0118] Freshly drawn venous blood (5 μl) was applied by automatic
pipette (Gilson) to the edge of the sample chamber. The progress of the
blood drawn into the sample chamber was recorded by a high-speed video
camera at a speed of 16 frames per second. Examples of captured video
images are shown in FIG. 4. Blood wicked into the sample chambers of the
biosensors containing mesh coated with surfactants FC-170C (control),
G2109, DC 193, DC 190, and FSN-100.

[0119] The purpose of this example was to qualitatively assess the
spreading ability of blood onto a nylon mesh coated with surfactants,
where the nylon mesh is incorporated into a biosensor having two layers
of mesh. The biosensors were stored 18 months prior to testing.

[0120] Biosensors of the type described in Example 3 were sealed in foil
packets and stored at ambient temperature for 18 months. The biosensors
were then tested with blood, and the average time of filling for the
sample chambers were determined from recorded video sequences (TABLE 5).

[0121] In each run, a sample of freshly drawn venous blood was applied by
automatic pipette (Gilson) to the edge of the sample chamber. The samples
of blood were drawn from two donors. Each sample contained 10 μl of
blood. For each type of surfactant, six biosensors were tested with blood
samples from one donor and six biosensors were tested with blood samples
from the other donor. The progress of the blood as it was drawn into the
sample chamber was recorded by a high-speed video camera at a speed of 16
frames per second.

[0122] Sample chambers containing mesh coated with the surfactant G2109
were not stable and failed to fill with blood. The best performance with
respect to filling was observed for the sample chambers containing mesh
coated with surfactant DC 193. The filling speed for the sample chambers
containing mesh coated with the surfactant DC 193 exceeded that of the
sample chambers containing mesh coated with the surfactant FC-170C.
Sample chambers utilizing mesh coated with the surfactant FSN-100
required almost twice as much time to fill as did sample chambers
utilizing mesh coated with surfactant DC 193. Sample chambers containing
mesh coated with the silicone surfactant DC 190 displayed filling
performance comparable to that of sample chambers containing mesh coated
with the surfactant FC-170C.

[0123] The purpose of this example was to qualitatively assess the
spreading ability of blood onto a polyester mesh, where the polyester
mesh is incorporated into a biosensor having one layer of mesh. The
biosensors were stored 18 months prior to testing.

[0124] Biosensors substantially similar to that described in WO 99/19507,
published 22 Apr. 1999, incorporated herein by reference, were
constructed, with the exception that the sample chamber of the biosensor
contained a single layer of polyester mesh coated with a surfactant. The
biosensors contained a working electrode, an electrode that performs as a
reference electrode and a counter electrode, and a trigger electrode. The
sample was introduced at the end of the biosensor strip, not through an
aperture in the cover layer. The layer of polyester mesh was PE130 mesh
supplied by Sefar (Switzerland). A dip coating method was used to coat
separate rolls of PE130 mesh with the various surfactants under
evaluation (TABLE 6). Aqueous isopropanol solutions of surfactants were
used for coating.

[0125] In each run, a sample of freshly drawn venous blood was applied by
automatic pipette (Gilson) to the edge of the sample chamber. The samples
of blood were drawn from two donors. Each sample contained 10 μl of
blood. For each type of surfactant, six biosensors were tested with blood
samples from one donor and six biosensors were tested with blood samples
from the other donor. The progress of the blood as it was drawn into the
sample chamber was recorded by a high-speed video camera at a speed of 16
frames per second.

[0126] Biosensors containing the surfactant G2109 were not stable and
failed to fill. The best performance with respect to filling was observed
for the sample chambers containing mesh coated with the surfactant DC
193. The filling speed for sample chambers containing mesh coated with
the surfactant DC 193 exceeded that of the sample chambers containing
mesh coated with the surfactant FC-170C. The sample chambers containing
mesh coated with the surfactant DC 190 exhibited greater filling speed as
compared with sample chambers containing mesh coated with the surfactant
FC-170C, but were deemed unsuitable on the ground of very poor precision
of electrode response. The surfactant DC 190 contains both EO and PO
whereas the surfactant DC 193 contains only EO (TABLE 1).

[0127] The purpose of this example was to quantitatively determine, by
infrared (IR) spectroscopy, the quantity of silicone surfactant DC 193
coated onto nylon and polyester mesh. The stability of a biosensor is
dependent upon the quantity of surfactant applied to the layer of mesh.

[0128] Various solutions of the surfactant DC 193 in water with
approximately 5% isopropylalcohol (depending on concentration of the
surfactant DC 193) were used as coating solutions. Polyester mesh (PE130,
roll width of approximately 1 m) was passed through the coating solution
and directed between two pinching rollers at a constant speed. The mesh
was then dried at a temperature of 120° C. After the side edges of
the roll of mesh were removed, the dried mesh was slit into rolls having
a width of 14 mm. The coating process was performed by Sefar
(Switzerland).

[0129] A length of polyester mesh (10 cm, PE130) was taken from a roll
(width of 14 mm) and cut into two five (5) cm lengths. Each sample was
weighed and then placed in a sealed glass test tube. Cyclohexane (5 ml;
minimum purity 99.8%) was added to the test tube, and the test tube
shaken for 15 minutes on an orbital shaker at a speed of 1400 rpm. The
resulting solution was tested by FT-IR using a liquid sample cell having
1 mm path length having barium fluoride windows. The scan conditions
were: 4 scans, 4 cm-1 resolution, 1200 to 1050 cm-1 range. The
total peak area was calculated for the absorbance spectrum from 1145 to
1080 cm-1. A calibration curve, obtained by measurement of known
concentrations of the surfactant DC 193 in cyclohexane, was used to
determine the quantity of the surfactant DC 193 in the extracted
solution, and, consequently, the coating weight of the surfactant on the
mesh sample.

[0130] The method described above can be used in a similar manner to
determine the coating weight of the surfactant DC 193 on the nylon meshes
NY64 and N151.

[0131] A linear relationship was observed between the concentration of
surfactant used in the coating solution and the weight of the surfactant
coated on the polyester mesh PE130 (see FIG. 5) and on the nylon meshes
NY64 (see FIG. 6) and NY151 (see FIG. 7), as determined by FT-IR and
expressed in terms of μg of surfactant per mg of mesh.

Example 7

[0132] The purpose of this example was to determine the frequency of blood
filling of biosensors having a single layer of mesh containing the
surfactant DC 193 coated thereon, as a function of concentration of
surfactant, storage temperature, and storage time.

[0133] Biosensors substantially similar to that described in Example 5
were used, except that the sample was introduced through an aperture
punched through the biosensor. Various coating weights were used. The
layer of polyester mesh was PE130 mesh, supplied by Sefar (Switzerland).
Separate rolls of PE130 mesh were dip coated in aqueous isopropanol
solutions of the surfactant DC 193.

[0134] The biosensors were packaged in foil and stored at ambient
temperature (22° C.), 30° C., 40° C., and 50°
C., for the purposes of the example. Ninety-six biosensors at each
concentration of the surfactant DC 193 and at each storage temperature
were tested at regular intervals to determine the percentage of the
biosensors that could be filled with blood. In each run, a sample of
freshly drawn venous blood was applied by automatic pipette (Gilson) to
the edge of the sample chamber. The samples of blood were drawn from two
donors. Each sample contained 10 μl of blood. Forty-eight biosensors
were tested with blood samples from one donor and forty-eight biosensors
were tested with blood samples from the other donor.

[0135] The stability profiles of the biosensors were determined by
plotting the percentage of the sample chambers of the biosensors that
filled against the square root of the storage period (see FIG. 8A).
Initially, 100% of the sample chambers filled with blood, but as storage
time increased, this percentage declined until it was not possible to
fill any sample chambers with blood. The stability profile can be
enhanced by increasing the coating concentration of the surfactant DC 193
and decreasing the storage temperature. This enhancement is clearly seen
in FIG. 8B. The shelf life required for the biosensor determines the
minimum amount of the surfactant DC 193 that must be coated onto the
layer of mesh. Biosensors containing polyester mesh coated with the
surfactant FC-170C exhibited significantly more stability than did those
biosensors having polyester mesh coated with the surfactant DC 193. For
example, no failure of biosensors having mesh coated with FC-170C
surfactant was observed at the storage temperature of 50° C. even
after one year of storage. However, such stability is considerably in
excess of that normally required (12 to 24 months at a storage
temperature of 30° C.), and this requirement is achieved by
coating the layer of mesh with the surfactant DC 193 at a sufficiently
high concentration. Shelf life of the biosensor for a requirement of 100%
filling at a storage temperature of 30° C. can be predicted from a
plot such as that shown in FIG. 9.

Example 8

[0136] The purpose of this example was to determine the time of filling
and the frequency of filling of biosensors having two layers of mesh in
the sample chamber as a function of the concentration of surfactant,
storage temperature, and storage time.

[0137] Biosensors of the type described in Example 3 were used. The sample
chambers of the biosensors contained two layers of nylon mesh, both
coated with the surfactant. The layers of nylon mesh were NY64 mesh and
NY151 mesh, both supplied by Sefar (Switzerland). Separate rolls of each
type of mesh were dip coated in a solution containing the surfactant DC
193. The concentration of the surfactant in the coating bath for the
NY151 mesh was 0.09% DC 193. For the NY64 mesh, the concentrations of the
surfactant in the coating bath were 0.35 and 1.05%.

[0138] The biosensors were packaged in foil and stored at ambient
temperature (22° C.), 30° C., 40° C., and 50°
C. for the purposes of the example. Ninety-six biosensors at each
concentration of DC 193 at each storage temperature were tested at
regular intervals to determine the percentage that can be filled with
blood. In each run, a sample of freshly drawn venous blood was applied by
automatic pipette (Gilson) to the edge of the sample chamber. The samples
of blood were drawn from two donors. Each sample contained 10 μA of
blood. Forty-eight biosensors were tested with blood samples from one
donor and forty-eight biosensors were tested with blood samples from the
other donor.

[0139] The stability profiles of the biosensors were assessed by plotting
the percentage of the sample chambers that filled and the time of filling
of the sample chambers as a function of the square root of storage time
(see FIGS. 10A, 10B, 11A, 11B). Initially, 100% of the sample chambers
filled with blood, but as the storage time increased, this percentage
declined. Testing was terminated at 52 weeks. Time of filling was
observed to increase as a function of the storage time and even more so
as the storage temperature increased. The stability of filling was
enhanced by increasing the coating concentration of the surfactant DC 193
and decreasing the storage temperature. Approximately 100% of the sample
chambers of the biosensors filled in under five (5) seconds when stored
at a temperature of 30° C. for a minimum of 12 months.

Example 9

[0140] The purpose of this example was to determine the time of filling of
a biosensor having a single layer of mesh in the sample chamber as a
function of concentration of surfactant and storage time at 50°
C., with a variety of silicone surfactants coated onto the layer of mesh.

[0141] Biosensors similar to that described in Example 5 were used. The
layer of polyester mesh was PE130 mesh supplied by Sefar (Switzerland). A
dip coating method was used to coat separate rolls of PE130 mesh with the
various surfactants being evaluated (TABLE 7). Aqueous isopropanol
solutions of surfactants at concentrations of 1% w/w and 3% w/w were used
for coating. A 50:50 mixture of the surfactants BC2213 and BC2234 was
also evaluated.

[0142] The biosensors were packaged in foil and stored at a temperature of
50° C. for the purposes of the example. A sample of freshly drawn
venous blood was applied by automatic pipette (Gilson) to the edge of the
sample chamber. The samples of blood were drawn from two donors. Each
sample contained 5 μl of blood. For each type of surfactant, six
biosensors were tested with blood samples from one donor and six
biosensors were tested with blood samples from the other donor. The
progress of the blood as it was drawn into the sample chamber was
recorded by a high-speed video camera at a speed of 16 frames per second.

[0150] The average time of filling was calculated for biosensors
containing different types of surfactants as a function of storage time
(0-12 weeks) at a temperature of 50° C. The resulting data is
shown in FIG. 12.

[0151] Stability failure points for the biosensors are indicated by large
increases in time of filling. Stability failure was observed for 1%
BC2213, 3% BC2213 and 1% BC2213 (50%)/BC2234 (50%). Increased stability
can be brought about by increasing the weight of surfactant on the mesh
layer (compare 3% BC2213 versus 1% BC2213). The stability of the silicone
surfactants follows the approximate order from the greatest to the
lowest:

[FC-170C (control)]>DC 193>C2234>BC2234 (50%)/BC2213
(50%)>BC2213

[0152] The low molecular weight trisiloxane surfactant BC2213 is the least
stable, but a mixture of BC2213 with BC2234 exhibits increased stability.

[0153] TABLE 8 compares initial time of filling for biosensors containing
the polyester mesh coated with various silicone surfactants with the
initial time of filling of biosensors containing the polyester mesh
coated with surfactant FC-170C. The time of filling follows the
approximate order from lowest to greatest:

BC2213>BC2234˜BC2234 (50%)/BC2213 (50%)>DC 193>FC-170C
(control)

[0154] The time of filling for all the sample chambers containing mesh
coated with silicone surfactants are lower than those for sample chambers
containing the mesh coated with the surfactant FC-170C (control). The
trend for time of filling is the converse of that seen for filling
stability. Mixtures of silicone surfactants can provide the best
compromise of filling speed and stability, e.g., a mixture of the
surfactants BC2213 (rapid filling and poor stability) and BC2234 or DC
193 (slow filling and good stability).

[0155] The purpose of this example was to qualitatively assess the
adhesion of a polyester film and an insulating ink to a layer of nylon
mesh coated with various surfactants in a biosensor having two layers of
mesh.

[0156] Biosensors of the type described in Example 3 were used. The sample
chambers of the biosensors contained two layers of nylon mesh, both
coated with a surfactant. The layers of nylon mesh were NY64 and NY151,
supplied by Sefar (Switzerland). The surfactants (G2109, DC 190, DC 193,
FSN-100 and FC-170C), listed in TABLE 4, were included in this example,
along with the anionic surfactant Aerosol OT-100 (listed in TABLE 1). The
biosensors were assessed for any compatibility or adhesion problems or
both between the various surfactants and any other materials.

[0157] In U.S. Pat. No. 5,628,890, the surfactant-coated mesh layer was
held in place by overprinting with a layer of insulating ink (Sericard,
commercially available from Sericol, Broadstairs, UK). The surfactants
listed in TABLE 4 (G2109, DC 190, DC 193, FSN-100 and FC-170C) were
compatible with the insulating ink. The polar anionic surfactant OT-100
was incompatible with the organic ink, with the result that it was not
possible to adhere mesh coated with that surfactant to the surface of the
electrode. For this reason, the surfactant OT-100 was rejected and no
further work was carried out with it.

[0158] The surfactant-coated mesh layer also came into contact with a top
layer of polyester film in those biosensors constructed according to U.S.
Pat. No. 5,628,890. It is necessary to provide good adhesion between the
polyester film and the surfactant-coated mesh where the sides of the
sample chamber of the biosensor are formed. If good adhesion is not
provided, the blood in the sample chamber may seep between the polyester
film and the mesh that is being held in place by the insulating ink at
the edge of the biosensor. The consequences of such seepage are that (1)
the volume of blood required by the biosensor will be increased, and (2)
the blood is not contained within the sample chamber, which may cause
trouble with handling. The surfactants listed in TABLE 4 (G2109, DC 190,
DC 193, and FC-170C), with the exception of FSN-100 provided biosensors
wherein the polyester film adhered sufficiently to the insulating layer
with little or no seepage of blood. The surfactant FSN-100 yielded
biosensors having poorly adhering polyester film, as demonstrated in
FIGS. 13A and 13B. The surfactant DC 193 performed well in this respect.

[0159] Various modifications and alterations of this invention will become
apparent to those skilled in the art without departing from the scope and
spirit of this invention, and it should be understood that this invention
is not to be unduly limited to the illustrative embodiments set forth
herein.